Production of syringyl lignin in gymnosperms

ABSTRACT

The present invention relates to a method for producing syringyl lignin in gymnosperms. The production of syringyl lignin in gymnosperms is accomplished by genetically transforming a gymnosperm genome, which does not normally contain genes which code for enzymes necessary for production of syringyl lignin, with DNA which codes for enzymes found in angiosperms associated with production of syringyl lignin. The expression of the inserted DNA is mediated using host promoter regions in the gymnosperm. In addition, genetic sequences which code for gymnosperm lignin anti-sense mRNA may be incorporated into the gymnosperm genome in order to suppress the formation of the less preferred forms of lignin in the gymnosperm such as guaiacyl lignin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/681,878, filed on Oct. 9, 2003, now U.S. Pat. No. 7,411,059, which isa continuation of U.S. patent application Ser. No. 09/796,256, filed onFeb. 28, 2001, now abandoned, which is a division of U.S. patentapplication Ser. No. 08/991,677, filed on Dec. 16, 1997, now U.S. Pat.No. 6,252,135, which claims the benefit of U.S. Provisional ApplicationNo. 60/033,381, filed Dec. 16, 1996.

FIELD OF THE INVENTION

The invention relates to the molecular modification of gymnosperms inorder to cause the production of syringyl units during ligninbiosynthesis and to production and propagation of gymnosperms containingsyringyl lignin.

BACKGROUND OF THE INVENTION

Lignin is a major part of the supportive structure of most woody plantsincluding angiosperm and gymnosperm trees which in turn are theprincipal sources of fiber for making paper and cellulosic products. Inorder to liberate fibers from wood structure in a manner suitable formaking many grades of paper, it is necessary to remove much of thelignin from the fiber/lignin network. Lignin is removed from wood chipsby treatment of the chips in an alkaline solution at elevatedtemperatures and pressure in an initial step of papermaking processes.The rate of removal of lignin from wood of different tree species variesdepending upon lignin structure. Three different lignin structures havebeen identified in trees: p-hydroxyphenyl, guaiacyl and syringyl, whichare illustrated in FIG. 1.

Angiosperm species, such as Liquidambar styraciflua L. [sweetgum], havelignin composed of a mixture of guaiacyl and syringyl monomer units. Incontrast, gymnosperm species such as Pinus taeda L. [loblolly pine] havelignin which is devoid of syringyl monomer units. Generally speaking,the rate of delignification in a pulping process is directlyproportional to the amount of syringyl lignin present in the wood. Thehigher delignification rates associated with species having a greaterproportion of syringyl lignin result in more efficient pulp milloperations since the mills make better use of energy and capitalinvestment and the environmental impact is lessened due to a decrease inchemicals used for delignification.

It is therefore an object of the invention to provide gymnosperm specieswhich are easier to delignify in pulping processes.

Another object of the invention is to provide gymnosperm species such asloblolly pine which contain syringyl lignin.

An additional object of the invention is to provide a method formodifying genes involved in lignin biosynthesis in gymnosperm species sothat production of syringyl lignin is increased while production ofguaiacyl lignin is suppressed.

Still another object of the invention is to produce whole gymnospermplants containing genes which increase production of syringyl lignin andrepress production of guaiacyl lignin.

Yet another object of the invention is to identify, isolate and/or clonethose genes in angiosperms responsible for production of syringyllignin.

A further object of the invention is to provide, in gymnosperms, geneswhich produce syringyl lignin.

Another object of the invention is to provide a method for making anexpression cassette insertable into a gymnosperm cell for the purpose ofinducing formation of syringyl lignin in a gymnosperm plant derived fromthe cell.

Definitions

The term “promoter” refers to a DNA sequence in the 5′ flanking regionof a given gene which is involved in recognition and binding of RNApolymerase and other transcriptional proteins and is required toinitiate DNA transcription in cells.

The term “constitutive promoter” refers to a promoter which activatestranscription of a desired gene, and is commonly used in creation of anexpression cassette designed for preliminary experiments relative totesting of gene function. An example of a constitutive promoter is 35SCaMV, available from Clonetech.

The term “expression cassette” refers to a double stranded DNA sequencewhich contains both promoters and genes such that expression of a givengene is achieved upon insertion of the expression cassette into a plantcell.

The term “plant” includes whole plants and portions of plants, includingplant organs (e.g. roots, stems, leaves, etc.)

The term “angiosperm” refers to plants which produce seeds encased in anovary. A specific example of an angiosperm is Liquidambar styraciflua(L.)[sweetgum]. The angiosperm sweetgum produces syringyl lignin.

The term “gymnosperm” refers to plants which produce naked seeds, thatis, seeds which are not encased in an ovary. A specific example of agymnosperm is Pinus taeda (L.)[loblolly pine]. The gymnosperm loblollypine does not produce syringyl lignin.

SUMMARY OF THE INVENTION

With regard to the above and other objects, the invention provides amethod for inducing production of syringyl lignin in gymnosperms and togymnosperms which contain syringyl lignin for improved delignificationin the production of pulp for papermaking and other applications. Inaccordance with one of its aspects, the invention involves cloning anangiosperm DNA sequence which codes for enzymes involved in productionof syringyl lignin monomer units, fusing the angiosperm DNA sequence toa lignin promoter region to form an expression cassette, and insertingthe expression cassette into a gymnosperm genome.

Enzymes required for production of syringyl lignin in an angiosperm areobtained by deducing an amino acid sequence of the enzyme, extrapolatingan mRNA sequence from the amino acid sequence, constructing a probe forthe corresponding DNA sequence and cloning the DNA sequence which codesfor the desired enzyme. A promoter region specific to a gymnospermlignin biosynthesis gene is identified by constructing a probe for agymnosperm lignin biosynthesis gene, sequencing the 5′ flanking regionof the DNA which encodes the gymnosperm lignin biosynthesis gene tolocate a promoter sequence, and then cloning that sequence.

An expression cassette is constructed by fusing the angiosperm syringyllignin DNA sequence to the gymnosperm promoter DNA sequence.Alternatively, the angiosperm syringyl lignin DNA is fused to aconstitutive promoter to form an expression cassette. The expressioncassette is inserted into the gymnosperm genome to transform thegymnosperm genome. Cells containing the transformed genome are selectedand used to produce a transformed gymnosperm plant containing syringyllignin.

In accordance with the invention, the angiosperm gene sequences bi-OMT,4CL, P450-1 and P-450-2 have been determined and isolated as associatedwith production of syringyl lignin in sweetgum and lignin promoterregions for the gymnosperm loblolly pine have been determined to be the5′ flanking regions for the 4CL1B, 4CL3B and PAL gymnosperm ligningenes. Expression cassettes containing sequences of selected genes fromsweetgum have been inserted into loblolly pine embryogenic cells andpresence of sweetgum genes associated with production of syringyl ligninhas been confirmed in daughter cells of the resulting loblolly pineembryogenic cells.

The invention therefore enables production of gymnosperms such asloblolly pine containing genes which code for production of syringyllignin, to thereby produce in such species syringyl lignin in the woodstructure for enhanced pulpability.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects of the invention will now be furtherdescribed in the following detailed specification considered inconjunction with the following drawings in which:

FIG. 1 illustrates a generalized pathway for lignin synthesis; and

FIGS. 2A-2E illustrate a bifunctional-O-methyl transferase (bi-OMT) genesequence involved in the production of syringyl lignin in an angiosperm(SEQ ID 5 coding SEQ ID 6);

FIGS. 3A-3G illustrate a 4-coumarate CoA ligase (4CL) gene sequenceinvolved in the production of syringyl lignin in an angiosperm (SEQ ID 7coding SEQ ID 8);

FIG. 4 illustrates a ferulic acid-5-hydroxylase (P450-1) gene sequenceinvolved in the production of syringyl lignin in an angiosperm (SEQ ID 1coding SEQ ID 2);

FIG. 5 illustrates a ferulic acid-5-hydroxylase (P450-2) gene sequenceinvolved in the production of syringyl lignin in an angiosperm (SEQ ID 3coding SEQ ID 4);

FIG. 6 illustrates nucleotide sequences of the 5′ flanking region of theloblolly pine 4CL1B gene showing the location of regulatory elements forlignin biosynthesis (SEQ ID 10);

FIGS. 7A-7B illustrate nucleotide sequences of the 5′ flanking region ofthe loblolly pine 4CL3B gene showing the location of regulatory elementsfor lignin biosynthesis (SEQ ID 11);

FIGS. 8A-8B illustrate nucleotide sequences of the 5′ flanking region ofloblolly pine PAL gene showing the location of regulatory elements forlignin biosynthesis (SEQ ID 9);

FIG. 9 illustrates a PCR confirmation of the sweetgum P450-1 genesequence in transgenic loblolly pine cells. Lanes 1-4: PCR amplificationof Sweetgum P450-1 gene from control and transgenic loblolly cell lines(Note the 600 by amplified fragment in lanes 2-4). Lanes 6-9: PCRamplification of Hygromycin gene from control and transgenic loblollypine cell lines (Note the 1000 by amplified fragment in lanes 7-9). Lane1: Control PT 52 line; Lane 2: Transgenic line Y2; Lane 3: Transgenicline Y17; Lane 4: Control plasmid pSSLsP450-1-iml-s; Lane 5: DNA sizemarker Phi 174/HaeII (BRL); top 4 bands indicate molecular size of 1354,1078, 872 and 603 bp; Lane 6: Control PT 52 line; Lane 7: Transgenicline Y7; Lane 8: Transgenic line 04; Lane 9: Control plasmid pHygro.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, a method is provided for modifying agymnosperm genome, such as the genome of a loblolly pine, so thatsyringyl lignin will be produced in the resulting plant, therebyenabling cellulosic fibers of the same to be more easily separated fromlignin in a pulping process. In general, this is accomplished by fusingone or more angiosperm DNA sequences (referred to at times herein as the“ASL DNA sequences”) which are involved in production of syringyl ligninto a gymnosperm lignin promoter region (referred to at times herein asthe “GL promoter region”) specific to genes involved in gymnospermlignin biosynthesis to form a gymnosperm syringyl lignin expressioncassette (referred to at times herein as the “GSL expression cassette”).Alternatively, the one or more ASL DNA sequences are fused to one ormore constitutive promoters to form a GSL expression cassette.

The GSL expression cassette preferably also includes selectable markergenes which enable transformed cells to be differentiated fromuntransformed cells. The GSL expression cassette containing selectablemarker genes is inserted into the gymnosperm genome and transformedcells are identified and selected, from which whole gymnosperm plantsmay be produced which exhibit production of syringyl lignin.

To suppress production of less preferred forms of lignin in gymnosperms,such as guaiacyl lignin, genes from the gymnosperm associated withproduction of these less preferred forms of lignin are identified,isolated and the DNA sequence coding for anti-sense mRNA (referred to attimes herein as the “GL anti-sense sequence”) for these genes isproduced. The DNA sequence coding for anti-sense mRNA is thenincorporated into the gymnosperm genome, which when expressed bind tothe less preferred guaiacyl gymnosperm lignin mRNA, inactivating it.

Further features of these and various other steps and proceduresassociated with practice of the invention will now be described in moredetail beginning with identification and isolation of ASL DNA sequencesof interest for use in inducing production of syringyl lignin in agymnosperm.

I. Determination of DNA Sequence for Genes Associated with Production ofSyringyl Lignin

The general biosynthetic pathway for production of lignin has beenpostulated as shown in FIG. 1. From FIG. 1, it can be seen that thegenes CCL, OMT and F5H (which is from the class of P450 genes) may playkey roles in production of syringyl lignin in some plant species, buttheir specific contributions and mechanisms remain to be positivelyestablished. It is suspected that the CCL, OMT and F5H genes may havespecific equivalents in a specific angiosperm, such as sweetgum.Accordingly, one aim of the present invention is to identify, sequenceand clone specific genes of interest from an angiosperm such as sweetgumwhich are involved in production of syringyl lignin and to thenintroduce those genes into the genome of a gymnosperm, such as loblollypine, to induce production of syringyl lignin.

Genes of interest may be identified in various ways, depending on howmuch information about the gene is already known. Genes believed to beassociated with production of syringyl lignin have already beensequenced from a few angiosperm species, viz, CCL and OMT.

DNA sequences of the various CCL and OMT genes are compared to eachother to determine if there are conserved regions. Once the conservedregions of the DNA sequences are identified, oligo-dT primers homologousto the conserved sequences are synthesized. Reverse transcription of theDNA-free total RNA which was purified from sweetgum xylem tissue,followed by double PCR using gene-specific primers, enables productionof probes for the CCL and OMT genes.

A sweetgum cDNA library is constructed in a host, such as lambda ZAPII,available from Stratagene, of LaJolla, Calif., using poly(A)+RNAisolated from sweetgum xylem, according to the methods described byBugos et al. (1995 Biotechniques 19:734-737). The above mentioned probesare used to assay the sweetgum cDNA library to locate cDNA which codesfor enzymes involved in production of syringyl lignin. Once a syringyllignin sequence is located, it is then cloned and sequenced according toknown methods which are familiar to those of ordinary skill.

In accordance with the invention, two sweetgum syringyl lignin geneshave been determined using the above-described technique. These geneshave been designated 4CL and bi-OMT. The sequence obtained for thesweetgum syringyl lignin gene, designated bi-OMT, is illustrated in FIG.2 (SEQ ID 5 and 6). The sequence obtained for the sweetgum syringyllignin gene, designated 4CL, is illustrated in FIG. 3 (SEQ ID 7 and 8).

An alternative procedure was employed to identify the F51-1 equivalentgenes in sweetgum. Because the DNA sequences for similar P450 genes fromother plant species were known, probes for the P450 genes were designedbased on the conserved regions found by comparing the known sequencesfor similar P450 genes. The known P450 sequences used for comparisoninclude all plant P450 genes in the GenBank database. Primers weredesigned based on two highly conserved regions which are common to allknown plant P450 genes. The primers were then used in a PCR reactionwith the sweetgum cDNA library as a template. Once P450-like fragmentswere located, they were amplified using standard PCR techniques, clonedinto a pBluescript vector available from Clonetech of Palo Alto, Calif.and transformed into a DH5.alpha. E. coli strain available from GibcoBRL of Gaithersburg, Md.

After E. coli colonies were tested in order to determine that theycontained the P450-like DNA fragments, the fragments were sequenced.Several P450-like sequences were located in sweetgum using the abovedescribed technique. One P450-like sequence was sufficiently differentfrom other known P450 sequences to indicate that it represented a newP450 gene family. This potentially new P450 cDNA fragment was used as aprobe to screen a full length clone from the sweetgum xylem library.These putative hydroxylase P450clones were designated P450-1 and P450-2.The sequence obtained for P450-1 and P450-2 are illustrated in FIG. 4(SEQ ID 1 and 2) and FIG. 5 (SEQ ID 3 and 4).

II. Identification of GL Gene Promoter Regions

In order to locate gymnosperm lignin promoter regions, probes aredeveloped to locate lignin genes. After the-gymnosperm lignin gene islocated, the portion of DNA upstream from the gene is sequenced,preferably using the GenomeWalker Kit, available from Clonetech. Theportion of DNA upstream from the lignin gene will generally contain thegymnosperm lignin promoter region.

Gymnosperm genes of interest include CCL-like genes and PAL-like genes,which are believed to be involved in the production of lignin ingymnosperms. Preferred probe sequences are developed based on previouslysequenced genes, which are available from the gene bank. The preferredgene bank accession numbers for the CCL-like genes include U39404 andU39405. A preferred gene bank accession number for a PAL-like gene isU39792. Probes for such genes are constructed according to methodsfamiliar to those of ordinary skill in the art. A genomic DNA library isconstructed and DNA fragments which code for gymnosperm lignin genes arethen identified using the above mentioned probes. A preferred DNAlibrary is obtained from the gymnosperm, Pinus taeda (L.) [LoblollyPine], and a preferred host of the genomic library is Lambda DashII,available from Stratagene of LaJolla, Calif.

Once the DNA fragments which code for the gymnosperm lignin genes arelocated, the genomic region upstream from the gymnosperm lignin gene(the 5′ flanking region) was identified. This region contains the GLpromoter. Three promoter regions were located from gymnosperm ligninbiosynthesis genes. The first is the 5′ flanking region of the loblollypine 4CL3B gene, shown in FIG. 6 (SEQ ID NO: 10). The second is the 5′flanking region of the loblolly pine gene 4CL1B, shown in FIG. 7 FIGS.7A-7B (SEQ ID NO: 11). The third is the 5′ flanking region of theloblolly pine gene PAL, shown in FIG. 8 (SEQ ID NO: 9).

III. Fusing the GL Promoter Region to the ASL DNA Sequence

The next step of the process is to fuse the GL promoter region to theASL DNA sequence to make a GSL expression cassette for insertion intothe genome of a gymnosperm. This may be accomplished by standardtechniques. In a preferred method, the GL promoter region is firstcloned into a suitable vector. Preferred vectors are pGEM7Z, availablefrom Promega, Madison, Wis. and SK available from Stratagene, ofLaJolla, Calif. After the promoter sequence is cloned into the vector,it is then released with suitable restriction enzymes. The ASL DNAsequence is released with the same restriction enzyme(s) and purified.

The GL promoter region sequence and the ASL DNA sequence are thenligated such as with T4 DNA ligase, available from Promega, to form theGSL expression cassette. Fusion of the GL and ASL DNA sequence isconfirmed by restriction enzyme digestion and DNA sequencing. Afterconfirmation of GL promoter-ASL DNA fusion, the GSL expression cassetteis released from the original vector with suitable restriction enzymesand used in construction of vectors for plant transformation.

IV. Fusing the ASL DNA Sequence to a Constitutive Promoter Region

In an alternative embodiment, a standard constitutive promoter may befused with the ASL DNA sequence to make a GSL expression cassette. Forexample, a standard constitutive promoter may be fused with P450-1 toform an expression cassette for insertion of P450-1 sequences into agymnosperm genome. In addition, a standard constitutive promoter may befused with P450-2 to form an expression cassette for insertion of P450-2into a gymnosperm genome. A constitutive promoter for use in theinvention is the double 35S promoter, available from Clonetech.

In the preferred practice of the invention using constitutive promoters,a suitable vector such as pBI221, is digested XbaI and HindIII torelease the 35S promoter. At the same time the vector pHygro, availablefrom International Paper, was digested by XbaI and HindIII to releasethe double 35S promoter. The double 35S promoter was ligated to thepreviously digested pBI221 vector to produce a new pBI221 with thedouble 35S promoter. This new pBI221 was digested with Sad and SmaI, torelease the GUS fragment. The vector is next treated with T4 DNApolymerase to produce blunt ends and the vector is self-ligated. Thisvector is then further digested with BamHI and XbaI, available fromPromega. After the pBI221 vector containing the constitutive promoterregion has been prepared, lignin gene sequences are prepared forinsertion into the pBI221 vector.

The coding regions of sweetgum P450-1 or P450-2 are amplified by PCRusing primer with restriction sites incorporated in the 5′ and 3′ ends.In one example, an XbaI site was incorporated at the 5 end and a BamHIsite was incorporated at the 3′ end of the sweetgum P450-1 or P450-2genes. After PCR, the P450-1 and P450-2 genes were separately clonedinto a TA vector available from Invitrogen. The TA vectors containingthe P450-1 and P450-2 genes, respectively, were digested by XbaI andBamHI to release the P450-1 or P450-2 sequences.

The p35SS vector, described above, and the isolated sweetgum P450-1 orP450-2 fragments were then ligated to make GLS expression cassettescontaining the constitutive promoter.

V. Inserting the Expression Cassette into the Gymnosperm Genome

There are a number of methods by which the GSL expression cassette maybe inserted into a target gymnosperm cell. One method of inserting theexpression cassette into the gymnosperm is by micro-projectilebombardment of gymnosperm cells. For example, embryogenic tissuecultures of loblolly pine may be initiated from immature zygoticembryos. Tissue is maintained in an undifferentiated state on semi-solidproliferation medium. For transformation, embryogenic tissue is s;suspended in liquid proliferation medium. Cells are then sieved through,a preferably 40 mesh screen, to separate small, densely cytoplasmiccells from large vacuolar cells.

After separation, a portion of the liquid cell suspension fraction isvacuum deposited onto filter paper and placed on semi-solidproliferation medium. The prepared gymnosperm target cells are thengrown for several days on filter paper discs in a petri dish.

A 1:1 mixture of plasmid DNA containing the selectable marker expressioncassette and plasmid DNA containing the P450-1 expression cassette maybe precipitated with gold to form microprojectiles. The microprojectilesare rinsed in absolute ethanol and aliqots are dried onto a suitablemacrocarrier such as the macrocarrier available from BioRad in Hercules,Calif.

Prior to bombardment, embryogenic tissue is preferably desiccated undera sterile laminar-flow hood. The desiccated tissue is transferred tosemi-solid proliferation medium. The prepared microprojectiles areaccelerated from the macrocarrier into the desiccated target cells usinga suitable apparatus such as a BioRad PDS-1000/HE particle gun. In apreferred method, each plate is bombarded once, rotated 180 degrees, andbombarded a second time. Preferred bombardment parameters are 1350 psirupture disc pressure, 6 mm distance from the rupture disc tomacrocarrier (gap distance), 1 cm macrocarrier travel distance, and 10cm distance from macrocarrier stopping screen to culture plate(microcarrier travel distance). Tissue is then transferred to semi-solidproliferation medium containing a selection agent, such as hygromycin B,for two days after bombardment.

Other methods of inserting the GSL expression cassette include use ofsilicon carbide whiskers, transformed protoplasts, Agrobacterium vectorsand electroporation.

VI. Identifying Transformed Cells

In general, insertion of the GSL expression cassette will typically becarried out in a mass of cells and it will be necessary to determinewhich cells harbor the recombinant DNA molecule containing the GSLexpression cassette. Transformed cells are first identified by theirability to grow vigorously on a medium containing an antibiotic which istoxic to non-transformed cells. Preferred antibiotics are kanamycin andhygromycin B. Cells which grow vigorously on antibiotic containingmedium are further tested for presence of either portions of the plasmidvector, the syringyl lignin genes in the GSL expression cassette; e.g.the angiosperm bi-OMT, 4CL, P450-1 or P450-2 gene, or by testing forpresence of other fragments in the GSL expression cassette. Specificmethods which can be used to test for presence of portions of the GSLexpression cassette include Southern blotting with a labeledcomplementary probe or PCR amplification with specific complementaryprimers. In yet another approach, an expressed syringyl lignin enzymecan be detected by Western blotting with a specific antibody, or byassaying for a functional property such as the appearance of functionalenzymatic activity.

VII. Production of a Gymnosperm Plant from the Transformed GymnospermCell

Once transformed embryogenic cells of the gymnosperm have beenidentified, isolated and multiplied, they may be grown into plants. Itis expected that all plants resulting from transformed cells willcontain the GSL expression cassette in all their cells, and that wood inthe secondary growth stage of the mature plant will be characterized bythe presence of syringyl lignin.

Transgenic embryogenic cells are allowed to replicate and develop into asomatic embryo, which are then converted into a somatic seedling.

VIII. Identification, Production and Insertion of a GL mRNA Anti-SenseSequence

In addition to adding ASL DNA sequences, anti-sense sequences may beincorporated into a gymnosperm genome, via GSL expression cassettes, inorder to suppress formation of the less preferred native gymnospermlignin. To this end, the gymnosperm lignin gene is first located andsequenced in order to determine its nucleotide sequence. Methods forlocating and sequencing amino acids which have been previously discussedmay be employed. For example, if the gymnosperm lignin gene has alreadybeen purified, standard sequencing methods may be employed to determinethe DNA nucleic acid sequence.

If the gymnosperm lignin gene has not been purified and functionallysimilar DNA or mRNA sequences from similar species are known, thosesequences may be compared to identify highly conserved regions and thisinformation used as a basis for the construction of a probe. Agymnosperm cDNA or genomic library can be probed with the abovementioned sequences to locate the gymnosperm lignin cDNA or genomic DNA.Once the gymnosperm lignin DNA is located, it may be sequenced usingstandard sequencing methods.

After the DNA sequence has been obtained for a gymnosperm ligninsequence, the complementary anti-sense strand is constructed andincorporated into an expression cassette. For example, the GL mRNAanti-sense sequence may be fused to a promoter region to form anexpression cassette as described above. In a preferred method, the GLmRNA anti-sense sequence is incorporated into the previously discussedGSL expression cassette which is inserted into the gymnosperm genome asdescribed above.

IX. Inclusion of Cytochrome P450 Reductase (CPR) to Enhance Biosynthesisof Syringyl Lignin in Gymnosperms

In the absence of external cofactors such as NADPH (an electron donor inreductive biosyntheses), certain angiosperm lignin genes such as theP450 genes may remain inactive or not achieve full or desired activityafter insertion into the genome of a gymnosperm. Inactivity orinsufficient activity can be determined by testing the resulting plantwhich contains the P450 genes for the presence of syringyl lignin insecondary growth. It is known that cytochrome P450 reductase (CPR) maybe involved in promoting certain reductive biochemical reactions, andmay activate the desired expression of genes in many plants.Accordingly, if it is desired to enhance the expression of theangiosperm syringyl lignin genes in the gymnosperm, CPR may be insertedin the gymnosperm genome. In order to express CPR, the DNA sequence ofthe enzyme is ligated to a constitutive promoter or, for a specificspecies such as loblolly pine, xylem-specific lignin promoters such asPAL, 4CL1B or 4CL3B to form an expression cassette. The expressioncassette may then be inserted into the gymnosperm genome by variousmethods as described above.

X. EXAMPLES

The following non-limiting examples illustrate further aspects of theinvention. In these examples, the angiosperm is Liquidambar styraciflua(L.)[sweetgum] and the gymnosperm is Pinus taeda (L.)[loblolly pine].The nomenclature for the genes referred to in the examples is asfollows:

Genes Biochemical Name 4CL (angiosperm) 4-coumarate CoA ligase bi-OMT(angiosperm) bifunctional-O-methyl transferase FA5HP450-1 (angiosperm)Cytochrome P450 P450-2 (angiosperm) Cytochrome P450 PAL (gymnosperm)phenylalanine ammonia-lyase 4CL1B (gymnosperm) 4-coumarate CoA ligase4CL3B (gymnosperm) 4-coumarate CoA ligase

Example 1 Isolating and Sequencing bi-OMT and 4CL Genes from anAngiosperm

A cDNA library for Sweetgum was constructed in Lambda ZAPII, availablefrom Stratagene, of LaJolla, Calif., using poly(A)+RNA isolated fromSweetgum xylem tissue. Probes for bi-OMT and 4CL were obtained throughreverse transcription of their mRNAs and followed by double PCR usinggene-specific primers which were designed based on the OMT and CCL cDNAsequences obtained from similar genes cloned from other species.

Three primers were used for amplifying OMT fragments. One was anoligo-dT primer. One was a bi-OMT, (which was used to clone genefragments through modified differential display technique, as describedbelow in Example 2) and the other two were degenerate primers, whichwere based on the conserved sequences of all known OMTs. The twodegenerate primers were derived based on the following amino acidsequences:

(SEQ ID 12) 5′-Gly Gly Met Ala Thr Tyr Cys Cys Ala Thr Thr Tyr Ala AlaCys Ala Ala Gly Gly Cys-3′ (primer #22) and (SEQ ID 13) 3′-Ala Ala AlaGly Ala Gly Ala Gly Asn Ala Cys Asn Asn Ala Asn Asn Ala Asn GlyAla-5′ (primer #23).

A 900 by PCR product was produced when oligo-dT primer and primer #22were used, and a 550 by fragment was produced when primer numbers 22 and23 were used.

Three primers were used for amplifying CCL fragments. They were derivedfrom the following amino acid sequences:

(SEQ ID 14) 5′-Thr Thr Gly Gly Ala Thr Cys Cys Gly Gly Ile Ala Cys IleAla Cys Ile Gly Gly Ile Tyr Thr Ile Cys Cys Ile Ala Ala Arg GlyGly-3′ (primer R1S) (SEQ ID 15) 5′-Thr Thr Gly Gly Ala Thr Cys Cys GlyThr Ile Gly Thr Ile Gly Cys Ile Cys Ala Arg Cys Ala Arg Gly Thr Ile GlyAla Tyr Gly Gly-3′ (primer H1S) and (SEQ ID 16) 3′-Cys Cys Ile Cys ThrTyr Thr Ala Asp Ala Cys Arg Thr Ala Asp Gly Cys Ile Cys Cys Ala Gly CysThr Gly Thr Ala-5′ (primer R2A)

R1S and H1S were both sense primers. Primer R2A was an anti-senseprimer. A 650 by fragment was produced if R1S and R2A primers were usedand a 550 by fragment was produced when primers H1S and R2A were used.The sequence of these three primers were derived from conservedsequences for plant CCLs.

The reverse transcription-double PCR cloning technique used for theseexamples consisted of adding 10 μgf DNA-free total RNA in 25μlDEPC-treated water to a microfuge tube. Next, the following solutionswere added:

a. 5× Reverse transcript buffer 8.0 μl,

b. 0.1 MDTT 4.0 μl

c. 10 mM dNTP 2.0 μl

d. 100 μM oligo-dT primers 8.0 μl

e. Rnasin 2.0 μl

f. Superscript II 1.0 μl

After mixing, the tube was incubated at a temperature of 42° C. for one(1) hour, followed by incubation at 70° C. for fifteen (15) minutes.Forty (40) μl of 1N NaOH was added and the tube was further incubated at68° C. for twenty (20) minutes. After the incubation periods, 800 of 1NHCl was added to the reaction mixture. At the same time, 17 μl NaOAc, 5μl glycogen and 768 μl of 100% ethanol were added and the reactionmixture was maintained at −80° C. for 15 minutes in order to precipitatethe cDNA. The precipitated cDNA was centrifuged at high speed at 4° C.for 15 minutes. The resulting pellet was washed with 70% ethanol andthen dried at room temperature, and then was dissolved in 20 μl ofwater.

The foregoing procedure produced purified cDNA which was used as atemplate to carry out first round PCR using primers #22 and oligo-dT forcloning OMT cDNA and primer R1S and R2A for cloning 4CL cDNA. For thefirst round PCR, a master mix of 50 μl for each reaction was prepared.Each 50 μl mixture contained:

a. 10× buffer 5 μl

b. 25 mM MgCl₂ 5 μl

c. 100 μM sense primer 1 μl (primer #22 for OMT and primer R1S for CCL).

d. 100 μl anti-sense primer 1 μl (oligo-dT primer for OMT and R2A forCCL).

e. 10 mM dNTP 1 μl

f. Tag. DNA polymerase 0.5 μl

Of this master mix, 48 μl was added into a PCR tube containing 2 μl ofcDNA for PCR. The tube was heated to 95° C. for 45 seconds, 52° C. forone minute and 72° C. for two minutes. This temperature cycle wasrepeated for 40 cycles and the mixture was then held at 72° C. for 10minutes.

The cDNA fragments obtained from the first round of PCR were used astemplates to perform the second round of PCR using primers 22 and 23 forcloning bi-OMT cDNA and primer H1S and R2A for cloning 4CL cDNA. Thesecond round of PCR conditions were the same as the first round.

The desired cDNA fragment was then subcloned and sequenced. After thesecond round of PCR, the product with the predicted size was excisedfrom the gel and ligated into a pUC19 vector, available from Clonetech,of Palo Alto, Calif., and then transformed into DH5.alpha., an E. colistrain, available from Gibco BRL, of Gaithersburg, Md. After the insertshad been checked for correct size, the colonies were isolated andplasmids were sequenced using a Sequenase kit available from USB, ofCleveland, Ohio. The sequences are shown in FIG. 2 (SEQ ID 5 and 6) andFIG. 3 (SEQ ID 7 and 8).

Example 2 Alternative Isolation Method of Angiosperm bi-OMT Gene

As previously mentioned, one bi-OMT clone was produced via modifieddifferential display technique. This method is another type of reversetranscription-PCR, in which DNA-free total RNA was reverse transcribedusing oligo-dT primers with a single base pair anchor to form cDNA. Theoligo-dT primers used for reverse transcription of mRNA to synthesizecDNA were:

4 T11A: TTTTTTTTTTTTTTA, (SEQ ID NO: 17) T11C: TTTTTTTTTTTTTTC, (SEQ IDNO: 18) and T11G: TTTTTTTTTTTTTTG, (SEQ ID NO: 19)

These cDNAs were then used as templates for radioactive PCR which wasconducted in the presence of the same oligo-dT primers as listed above,a bi-OMT gene-specific primer and 35S-dATP. The OMT gene-specific primerwas derived from the following amino acid sequence:

(SEQ ID NO: 20) 5′-Cys Cys Asn Gly Gly Asn Gly Gly Ser Ala Arg GlyAla-3′.

The following PCR reaction solutions were combined in a microfuge tube:

a. H₂O 9.2 μl,

b. Taq Buffer 2.0 μl

c. dNTP (25 μM) 1.6 μl

d. Primers (5 μM) 2 μl, for each primer

e. ³⁵S-dATP 1 μl

f. Taq. pol. 0.2 μl

g. cDNA 2.0 μl.

The tube was heated to a temperature of 94° C. and held for 45 seconds,then at 37° C. for 2 minutes and then 72° C. for 45 seconds for fortycycles, followed by a final reaction at 72° C. for 5 minutes.

The amplified products were fractionated on a denaturing polyacrylamidesequencing gel and autoradiography was used to identify and excise thefragments with a predicted size. The designed OMT gene-specific primerhad a sequence conserved in a region toward the 3′-end of the OMT cDNAsequence. This primer, together with oligo-dT, was amplified into a OMTcDNA fragment of about 300 bp.

Three oligo-dTs with a single base pair of A, C or G, respectively, wereused to pair with the OMT gene-specific primer. Eight potential OMT cDNAfragments with predicted sizes of about 300 bp were excised from thegels after several independent PCR rounds using different combinationsof oligo-dT and OMT gene-specific oligo-nucleotides as primers.

The OMT cDNA fragments were then re-amplified. A Southern blot analysiswas performed for the resulting cDNAs using a 360 base-pair, ³²Pradio-isotope labeled, aspen OMT cDNA 3′-end fragment as a probe toidentify the cDNA fragments having a strong hybridization signal, underlow stringency conditions. Eight fragments were identified. Out of theseeight cDNA fragments, three were selected based on their highhybridization signal for sub-cloning and sequencing. One clone,LsOMT3′-1, (where the “Ls” prefix indicates that the clone was derivedfrom the Liquidambar styraciflua (L.) genome) was confirmed to encodebi-OMT based on its high homology to other lignin-specific plant OMTs atboth nucleotide and amino acid sequence levels.

A cDNA library was constructed in Lambda ZAP II, available fromStratagene, of LaJolla, Calif., using 5 mg poly(A)+RNA isolated fromsweetgum xylem tissue. The primary library consisting of approximately0.7×10⁶ independent recombinants was amplified and approximately 10⁵plaque-forming-units (pfu) were screened using a homologous 550base-pair probe. The hybridized filter was washed at high stringency(0.25×SSC, 0.1% SDS, 65° C.) conditions. The colony containing thebi-OMT fragment identified by the probe was eluted and the bi-OMTfragment was produced. The sequence as illustrated in FIG. 2 (SEQ ID 5and 6) was obtained.

Example 3 Isolating and Producing the DNA which Codes for the AngiospermP450-1 Gene

In order to find putative P450 cDNA fragments as probes for cDNA libraryscreening, a highly degenerated sense primer based on the amino acidsequence of 5′-Glu, Glu, Phe, Arg, Pro, Glu, Arg-3′ was designed basedon the conserved regions found in some plant P450 proteins. Thisconserved domain was located upstream of another highly conserved regionin P450 proteins, which had an amino acid sequence of 5′-Phe Gly Xaa GlyXaa Xaa Cys Xaa Gly-3′ (SEQ ID 21). This primer was synthesized with theincorporation of an XboI restriction site to give a 26-base-pairoligomer with a nucleotide sequence of 5′ ATG TGC AGT TTT TTT TTT TTTTIT TT-3′ (SEQ ID 22).

This primer and the oligo-dT-XhoI primer were then used to perform PCRreactions with the sweetgum cDNA library as a template. The cDNA librarywas constructed in Lambda ZAPII, available from Stratagene, of LaJolla,Calif., using poly(a)+RNA isolated from Sweetgum xylem tissue. Amplifiedfragments of 300 to 600 by were obtained. Because the designed primerwas located upstream of the highly conserved P450 domain, this designdistinguished whether the PCR products were P450 gene fragmentsdepending on whether they contained the highly conserved amino aciddomain.

All the fragments obtained from the PCR reaction were then cloned into apUC19 vector, available from Stratagene, of LaJolla, Calif., andtransformed into a DH5.alpha. E. coli strain, available from Gibco BRL,of Gaithersburg, Md.

Twenty-four positive colonies were obtained and sequenced. Sequenceanalysis indicated four groupings within the twenty-four colonies. Onewas C4H, one was an unknown P450 gene, and two did not belong to P450genes. Homologies of P450 genes in different species are usually morethan 80%. Because the homologies between the P450 gene families foundhere were around 40%, the sequence analysis indicated that a new P450gene family was sequenced. Moreover, since this P450 cDNA was isolatedfrom xylem tissue, it was highly probable that this P450 gene wasP450-1.

The novel sweetgum P450 cDNA fragment was used as a probe to screen afull length cDNA encoding for P450-1. Once the P450-1 gene was locatedit was sequenced. The length of the P450-1 cDNA is 1707 by and itcontains 45 by of 5′ non-coding region and 135 by of 3′ non-codingregion. The deduced amino acid sequence also indicates that this P450cDNA has a hydrophobic core at the N-terminal, which could be regardedas a leader sequence for c-translational targeting to membranes duringprotein synthesis. At the C-terminal region, there is a heme bindingdomain that is characteristic of all P450 genes. The P450-1 sequence, asillustrated in FIG. 4 (SEQ ID 1 and 2), was produced, according to theabove described methods.

Example 4 Isolating and Producing the DNA which Codes for the AngiospermP450-2 Gene

By using similar strategy of synthesizing PCR primers from the publishedliterature for hydroxylase genes in plants, another full length P450cDNA has been isolated that shows significant similarity with a putitiveF5H clone from Arabidopsis (Meyers et al. 1996: PNAS 93, 6869-6874).This cloned cDNA, designated P450-2, contains 1883 by and encodes anopen reading frame of 511 amino acids. The amino acid similarity sharedbetween Arabidopsis FSH and the P450-2 sweetgum clone is about 75%.

To confirm the function of the P450-2 gene, it was expressed in E. coli,strain, DH5 alpha, via pQE vector preparation, according to directionsavailable with the kit. A CO—Fe2+binding assay was also performed toconfirm the expression of P450-2 as a functional P450 gene. (Omura &Sato 1964, J. of Biochemistry 239: 2370-2378, Babriac et. al. 1991Archives of Biochemistry and Biophysics 288:302-309). The CO—Fe2+binding assay showed a peak at 450 nm which indicates that P450-2 hasbeen overexpressed as a functional P450 gene.

The P450-2 protein was further purified for production of antibodies inrabbits, and antibodies have been successfully produced. In addition,Western blots show that this antibody is specific to the membranefraction of sweetgum and aspen xylem extract. When the P450-2 antibodywas added to a reaction mixture containing aspen xylem tissue, enzymeinhibition studies showed that the activity of P450 in aspen was reducedmore than 60%, a further indication that P450-2 performs a p450likefunction. Recombinant P450-2 protein co-expressed with Arabidopsis CPRprotein in a baculovirus expression system hydroxylated ferulic acid(specific activity: 7.3 pKat/mg protein), cinnaminic acid (specificactivity: 25 pKat/mg protein, and p-coumeric acid (specific activity 3.8pKat/ng protein). The P450-2 enzyme which may be referred to as C4C3F5-Happears to be a broad spectrum hydroxylase in the phenyproponoid pathwayin plants FIG. 5 (SEQ ID 3 and 4) illustrates the P450-2 sequence.

Example 5 Identifying Gymnosperm Promoter Regions

In order to identify gymnosperm promoter regions, sequences fromloblolly pine PAL and CL1B and 4CL3B lignin genes were used as primersto screen the loblolly pine genomic library, using the GenomeWalker Kit.The loblolly pine PAL primer sequence was obtained from the GenBank,reference number U39792. The loblolly pine 4CL1B primer sequences werealso obtained from the gene bank, reference numbers U39404 and U39405.

The loblolly pine genomic library was constructed in Lambda DashII,available from Stratagene, of LaJolla, Calif. 3×10⁶ phage plaques fromthe genomic library of loblolly pine were screened using both the abovementioned PAL cDNA and 4CL (PCR clone) fragments as probes. Five 4CLclones were obtained after screening. Lambda DNAs of two 4CL of the five4CL clones obtained after screening were isolated and digested by EcoRV,Pstd, SalI and XbaI for Southern analysis. Southern analysis using 4CLfragments as probes indicated that both clones for the 4CL gene wereidentical. Results from further mapping showed that none of the originalfive 4CL clones contained promoter regions. When tested, the PAL clonesobtained from the screening also did not contain promoter regions.

In a second attempt to clone the promoter regions associated with thePAL and 4CL a Universal GenomeWalker™ kit, available from CLONETECH, wasused. In the process, total DNA from loblolly pine was digested byseveral restriction enzymes and ligated into the adaptors (libraries)provided with the kit. Two gene-specific primers for each gene weredesigned (GSP1 and 2). After two rounds of PCR using these primers andadapter primers of the kit, several fragments were amplified from eachlibrary. A 1.6 kb fragment and a 0.6 kb fragment for PAL gene and a 2.3kb fragment (4CL1B) and a 0.7 kb fragment (4CL3B) for the 4CL gene werecloned, sequenced and found to contain promoter regions for all threegenes. See FIG. 6 (SEQ ID 10), 7 (SEQ ID 11) and 8 (SEQ ID 9).

Example 6 Fusing the ASL DNA Sequence to a Constitutive Promoter Regionand Inserting the Expression Cassette Into a Gymnosperm Genome

As a first step, a ASL DNA sequence, P450-1, was fused with aconstitutive promoter region according to the methods described in theabove Section IV to form an P450-1 expression cassette. A second ASL DNAsequence, P450-2, was then fused with a constitutive promoter in thesame manner to form an P450-2 expression cassette. The P450-1 expressioncassette was inserted into the gymnosperm genome by micro-projectilebombardment. Embryogenic tissue cultures of loblolly pine were initiatedfrom immature zygotic embryos. The tissue was maintained in anundifferentiated state on semi-solid proliferation medium, according tomethods described by Newton et al. TAES Technical Publication “SomaticEmbryogenesis in Slash Pine”, 1995 and Keinonen-Mettala et al. 1996,Scand. J. For. Res. 11: 242-250.

After separation, 5 ml of the liquid cell suspension fraction whichpasses through the 40 mesh screen was vacuum deposited onto filter paperand placed on semi-solid proliferation medium. The prepared gymnospermtarget cells were then grown for 2 days on filter paper discs placed onsemi-solid proliferation medium in a petri dish. These target cell werethen bombarded with plasmid DNA containing the P450-1 expressioncassette and an expression cassette containing a selectable marker geneencoding the enzyme which confers resistance to the antibiotichygromycin B. A 1:1 mixture of selectable marker expression cassette andplasmid DNA containing the P450-1 expression cassette is precipitatedwith gold (1.5-3.0 microns) as described by Sanford et al. (1992). TheDNA-coated microprojectiles were rinsed in absolute ethanol and aliquotsof 10 μl (5 μg DNA/3 mg gold) were dried onto a macrocarrier, such asthose available from BioRad (Hercules, Calif.).

Prior to bombardment, embryogenic tissue was desiccated under a sterilelaminar-flow hood for 5 minutes. The desiccated tissue was transferredto semi-solid proliferation medium. The microprojectiles wereaccelerated into desiccated target cells using a BioRad PDS-1000/HEparticle gun.

Each plate was bombarded once, rotated 180 degrees, and bombarded asecond time. Preferred bombardment parameters were 1350 psi rupture discpressure, 6 mm distance from the rupture disc to macrocarrier (gapdistance), 1 cm macrocarrier travel distance, and 10 cm distance frommacrocarrier stopping screen to culture plate (microcarrier traveldistance). Tissue was then transferred to semi-solid proliferationmedium containing hygromycin B for two days after bombardment.

The P450-2 expression cassette was inserted into the gymnosperm genomeaccording to the same procedures.

Example 7 Selecting Transformed Target Cells

After insertion of the P450-1 expression cassette and the selectablemarker expression cassette into the gymnosperm target cells as describedin Example 6, transformed cells were selected by exposure to anantibiotic that causes mortality of any cells not containing the GSLexpression cassette. Forty independent cell lines were established fromcultures cobombarded with an expression cassette containing a hygromycinresistance gene construct and the P450-1 construct. These cell linesinclude lines Y2, Y17, Y7 and 04, as discussed in more detail below.

PCR techniques were then used to verify that the P450-1 gene had beensuccessfully integrated into the genomes of the established cell linesby extracting genomic DNA using the Plant DNAeasy kit, available fromQuaigen. 200 ng DNA from each cell line were used for each PCR reaction.Two P450-1 specific primers were designed to perform a PCR reaction witha 600 by PCR product size. The primers were:

(SEQ ID NO: 23) LsP450-iml-S primer: ATGGCTTTCCTTCTAATACCCATCTC, and(SEQ ID NO: 24) LsP450-iml-A primer: GGGTGTAATGGACGAGCAAGGACTTG.

Each PCR reaction (100 μl) consisted of 75 μl H2O, 1 μl MgCl (25 mM), 10μl PCR buffer 1 μl 10 mM dNTPs, and 10 μl DNA. 100 μl oil was layered onthe top of each reaction mix. Hot start PCR was done as follows: PCRreaction was incubated at 95 degrees C. for 7 minutes and 1 μl each ofboth LsP450-im1-S and LsP450-im1-A primers (100 μM stock) and 1 μl ofTaq polymerase were added through oil in each reaction. The PCR programused was 95 degrees C. for 1.5 minutes, 55 degrees C. for 45 sec and 72degrees C. for 2 minutes, repeated for 40 cycles, followed by extensionat 72 degrees C. for 10 minutes.

The above PCR products were employed to determine if gymnosperm cellscontained the angiosperm lignin gene sequences. With reference to FIG.9, PCR amplification was performed using template DNA from cells whichgrew vigorously on hygromycin B-containing medium. The PCR products wereelectrophoresed in an agarose gel containing 9 lanes. Lanes 14 containedPCR amplification of products of the Sweetgum P450-1 gene from anon-transformed control and transgenic loblolly pine cell lines. Lane 1contained the non-transformed control PT52. Lane 2 contained transgenicline Y2. Lane 3 contained transgenic line Y17 and Lane 4 contained theplasmid which contains the expression cassette pSSLsP450′-im-s. Lanes 2through 4 all contain an amplified fragment of about 600 bp, indicatingthat the P450-1 gene has been successfully inserted into transgenic celllines Y2 and Y17.

Lane 5 contained a DNA size marker Phi 174/HaeII (BRL). The top fourbands in this lane indicate molecular sizes of 1353, 1078, 872 and 603bp.

Lanes 6-9 contained PCR amplification products of hygromycin B gene fromnon-transformed control and transgenic loblolly pine cell lines. Lane 6contained the non-transformed control lane referenced to as PTS. Lane 7contained transgenic line Y7. Lane 8 contained transgenic line 04. Lane9 contained the plasmid which includes the expression cassettecontaining the gene encoding the enzyme which confers resistance to theantibiotic hygromycin B. Lanes 7-9 all show an amplified fragment ofabout 1000 bp, indicating that the hygromycin gene has been successfullyinserted into transgenic lines Y7 and 04.

These PCR results confirmed the presence of P450-1 and hygromycinresistance gene in transformed loblolly pine cell cultures. The resultsobtained from the PCR verification of 4 cell lines, and similar testswith the remaining 36 cell lines, confirm stable integration of theP450-1 gene and the hygromycin B gene in 25% of the 40 cell lines.

In addition, loblolly pine embryogenic cells which have beenco-bombarded with the P450-2 and hygromycin B expression cassettes, aregrowing vigorously on hygromycin selection medium, indicating that theP450-2 expression cassette was successfully integrated into thegymnosperm genome.

Although various embodiments and features of the invention have beendescribed in the foregoing detailed description, those of ordinary skillwill recognize the invention is capable of numerous modifications,rearrangements and substitutions without departing from the scope of theinvention as set forth in the appended claims. For example, in the casewhere the lignin DNA sequence is transcribed and translated to produce afunctional syringyl lignin gene, those of ordinary skill will recognizethat because of codon degeneracy a number of polynucleotide sequenceswill encode the same gene. These variants are intended to be covered bythe DNA sequences disclosed and claimed herein. In addition, thesequences claimed herein include those sequences with encode a genehaving substantial functional identity with those claimed. Thus, in thecase of syringyl lignin genes, for example, the DNA sequences includevariant polynucleotide sequences encoding polypeptides which havesubstantial identity with the amino acid sequence of syringyl lignin andwhich show syringyl lignin activity in gymnosperms.

1. An isolated DNA sequence comprising the promoter of SEQ ID NO:11.